The harvest of cells from tissue for maintenance and propagation in vitro by tissue culture is a major tool in medical and biochemical research. Tissue culture is the technique or process of proliferating and/or supporting the metabolism of tissues or cells derived from organisms (plant or animal) in a formulated nutritive environment. Once isolated by gentle tissue dissociation, cells are incubated in nutritive media capable of supporting life functions. With few exceptions, cells require attachment to a substratum in order to perform normal metabolic functions, grow and divide. In tissue, the substratum, which provides the support for cell growth, is either the basement membrane or interstitial matrix. Basement membranes act not only as physical scaffolds and molecular filters, but also as solid-phase regulators of a variety of cellular processes, including attachment, motility and differentiation. Basement membranes can also modulate cellular growth by acting as a reservoir for growth factors and by prolonging their in vivo half-life.
The basement membrane is a specific type of extracellular matrix and is composed primarily of laminin and type IV collagen. The four major matrix components characteristic of basement matrices include laminin, collagen IV, entactin, nidogen and heparan sulfate proteoglycans (HSPG). The basement membrane also contains a number of growth factors, such as EGF, IGF-1, PDGF, TGF-beta, VEGF, and bFGF. Examples of commercially available basement membrane-derived extracellular matrices include, for example, ECM gel by Sigma-Aldrich, or Matrigel® by Becton, Dickinson & Company, which is extracted from the Engelbreth-Holm-Swarm (EHS) mouse tumor. This mouse tumor is rich in basement membrane proteins. At room temperature, Matrigel® Matrix gels to form reconstituted basement membrane and is similar in its structure, composition, physical property and ability to retain functional characteristics typical of basement membranes in vivo.
Matrigel® matrix supports differentiation of many cell types, including endothelial cells, epithelial cells, neurons and hepatocytes. When endothelial cells are plated on Matrigel®, the cells stop proliferating, display high motility and cell-cell communication. Furthermore, the cells align and form a three-dimensional network of tube or duct-like structures. The formation of these structures has been used as a model of endothelial cell differentiation, as well as the final step of the angiogenic cascade.
The use of Matrigel® in endothelial cell tube formation assays is one of the major in vitro applications of Matrigel® matrix. In these assays, the extent of endothelial cell tube length is measured and used as an indicator of endothelial cell tube formation. On typical extracellular matrix cell differentiation, cell tube formation can occur even in the absence of exogenously added growth factors due to the presence of growth factors in the matrix. This has limited the application of the extracellular matrix to studies involving inhibitory effects. In particular, it has been difficult to study stimulatory effects because the background tube formation is too high. As a result, tube formation assays have thus far not been sensitive enough for stimulation studies.
It is thus, one object of the present invention to provide a modified reconstituted basement membrane-derived extracellular matrix composition to reduce background cell tube formation, in the absence of added growth factors, by altering the matrix structure and/or by sequestering or blocking accessibility of the growth factors in the composition to cells.
Another object of the present invention is to provide a cell culturing system that allows direct assessment of potential angiogenesis stimulators, as well as inhibitors for their effects on cell tube formation of endothelial cells, and other cell types. It would also be beneficial to provide an assay system for studying action pathways of these stimulatory and inhibitory agents.
Additionally, an object of the present invention is to provide an assay system which is rapid and cost-effective, and which significantly increases the signal dynamic range.